Carbamates of rapamycin

ABSTRACT

A compound of the structure ##STR1## wherein R 1  and R 2  are each, independently, hydrogen, --CONH--[(CR 3  R 4 ) m  (--A--(CR 5  R 6 ) n ) p  ] q  --B; ##STR2## R 3 , R 4 , R 5 , R 6 , and B are each, independently, hydrogen, alkyl, alkenyl, alkynyl, hydroxyalkyl, alkoxyalkyl, alkylthioalkyl, alkylaminoalkyl, dialkylaminoalkyl, arylalkyl, cycloalkyl, --OR 7 , --SR 7 , halogen, --CN, --NO 2 , --CF 3 , --COR 7 , --CO 2  R 7 , --CONHR 7 , --SO 2  R 7 , --OSO 3  R 7 , --NR 7  R 8 , --NHCOR 7 , --NHSO 2  R 7 , or Ar; 
     R 7  and R 8  are each, independently, hydrogen, alkyl, arylalkyl, alkenyl, alkynyl, hydroxyalkyl, alkoxyalkyl, alkylthioalkyl, alkylaminoalkyl, dialkylaminoalkyl, cycloalkyl; 
     R 9  and R 10  are each, independently, alkyl , alkenyl, alkynyl, hydroxyalkyl, alkoxyalkyl, alkylthioalkyl, alkylaminoalkyl, dialkylaminoalkyl, arylalkyl, --CF 3 , --COR 7 , --CO 2  R 7 , --CONHR 7 , --SO 2  R 7 , or Ar; 
     A is --CH 2  --, --NR 7 , --O--, --S--, --SO--, --SO 2  --, --PR 7 , --CO--, --NHCO--, --NHSO--, or --P(O)(R 7 )--; 
     Ar is phenyl, naphthyl, pyridyl, quinolyl, isoquinolyl, quinoxalyl, thienyl, thionaphthyl, furyl, benzofuryl, benzodioxyl, benzoxazolyl, benzoisoxazolyl, indolyl, thiazolyl, isoxazolyl, pyrimidinyl, pyrazinyl, imidazolyl, benzopyranyl, benz[b]thiophenolyl, benzimidazolyl, benzthiazolyl, benzodioxolyl, piperidinyl, morpholinyl, piperazinyl, tetrahydrofuranyl, or pyrrolidinyl; wherein the Ar group may be optionally mono-, di-, or tri-substituted; ##STR3##  is a nitrogen containing heterocycle that may be saturated, unsaturated, or partially unsaturated, and may be optionally mono-, di-, or tri- substituted; with the proviso that R 1  and R 2  are not both hydrogen; 
     m=0-6; 
     n=0-6; 
     p=0-1; 
     q=0-1; 
     or a pharmaceutically acceptable salt thereof which is useful as an immunosuppressive, antiinflammatory, antifungal, antiproliferative, and antitumor agent.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a division of U.S. patent application Ser. No. 08/297,663, filedSep. 1, 1994, (now U.S. Pat. No. 5,480,989) which is acontinuation-in-part of U.S. patent application Ser. No. 08/160,984,filed Dec. 1, 1993, now abandoned, which is a divisional of U.S. patentapplication Ser. No. 08/054,655, filed Apr. 23, 1993 (now U.S. Pat. No.5,302,584), which is a continuation-in-part of U.S. Patent applicationSer. No. 07/960,597, filed Oct. 13, 1992, now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to carbamates of rapamycin and a method for usingthem for inducing immunosuppression, and in the treatment oftransplantation rejection, host vs. graft disease, autoimmune diseases,diseases of inflammation, solid tumors, fungal infections, andhyperproliferative vascular disorders.

Rapamycin is a macrocyclic triene antibiotic produced by Streptomyceshygroscopicus, which was found to have antifungal activity, particularlyagainst Candida albicans, both in vitro and in vivo [C. Vezina et al.,J. Antibiot. 28, 721 (1975); S. N. Sehgal et al., J. Antiblot. 28, 727(1975); H. A. Baker et al., J. Antiblot. 31,539 (1978); U.S. Pat. No.3,929,992; and U.S. Pat. No. 3,993,749].

Rapamycin alone (U.S. Pat. No. 4,885,171) or in combination withpicibanil (U.S. Pat. No. 4,401,653) has been shown to have antitumoractivity. R. Martel et al. [Can. J. Physiol. Pharmacol. 55, 48 (1977)]disclosed that rapamycin is effective in the experimental allergicencephalomyelitis model, a model for multiple sclerosis; in the adjuvantarthritis model, a model for rheumatoid arthritis; and effectivelyinhibited the formation of IgE-like antibodies.

The immunosuppressive effects of rapamycin have been disclosed in FASEB3, 3411 (1989). Cyclosporin A and FK-506, other macrocyclic molecules,also have been shown to be effective as immunosuppressive agents,therefore useful in preventing transplant rejection [FASEB 3, 3411(1989); FASEB 3, 5256 (1989); R. Y. Calne et al., Lancet 1183 (1978);and U.S. Pat. No. 5,100,899].

Rapamycin has also been shown to be useful in preventing or treatingsystemic lupus erythematosus [U.S. Pat. No. 5,078,999], pulmonaryinflammation [U.S. Pat. No. 5,080,899], insulin dependent diabetesmellitus [Fifth Int. Conf. Infiamm. Res. Assoc. 121 (Abstract), (1990)],and smooth muscle cell proliferation and intimal thickening followingvascular injury [Morris, R. J. Heart Lung Transplant 11 (pt. 2): 197(1992)].

Mono- and diacylated derivatives of rapamycin (esterified at the 28 and43 positions) have been shown to be useful as antifungal agents (U.S.Pat. No. 4,316,885) and used to make water soluble prodrugs of rapamycin(U.S. Pat. No. 4,650,803). Recently, the numbering convention forrapamycin has been changed; therefore according to Chemical Abstractsnomenclature, the esters described above would be at the 31- and42-positions. U.S. Pat. No. 5,118,678 discloses carbamates of rapamycinthat are useful as immunosuppressive, anti-inflammatory, antifungal, andantitumor agents.

DESCRIPTION OF THE INVENTION

This invention provides derivatives of rapamycin which are useful asimmunosuppressive, antiinflammatory, antifungal, antiproliferative, andantitumor agents having the structure ##STR4## wherein R¹ and R² areeach, independently, hydrogen, --CONH--[(CR³ R⁴)_(m) (--A--(CR⁵R⁶)_(n))_(p) ]_(q) --B; ##STR5## R³, R⁴, R⁵, R⁶, and B are each,independently, hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7carbon atoms, alkynyl of 2-7 carbon atoms, hydroxyalkyl of 1-6 carbonatoms, alkoxyalkyl of 2-12 carbon atoms, alkylthioalkyl of 2-12 carbonatoms, alkylaminoalkyl of 2-12 carbon atoms, dialkylaminoalkyl of 3-12carbon atoms, arylalkyl of 7-10 carbon atoms, cycloalkyl of 3-8 carbonatoms, --OR⁷, --SR⁷, halogen, --CN, --NO₂, --CF₃, --COR⁷, --CO₂ R⁷,--CONHR⁷, --SO₂ R⁷, --OSO₃ R⁷, --NR⁷ R⁸, --NHCOR⁷, --NHSO₂ R⁷, or Ar;

R⁷ and R⁸ are each, independently, hydrogen, alkyl of 1-6 carbon atoms,arylalkyl of 7-10 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of2-7 carbon atoms, hydroxyalkyl of 1-6 carbon atoms, alkoxyalkyl of 2-12carbon atoms, alkylthioalkyl of 2-12 carbon atoms, alkylaminoalkyl of2-12 carbon atoms, dialkylaminoalkyl of 3-12 carbon atoms, cycloalkyl of3-8 carbon atoms, or Ar;

R⁹ and R¹⁰ are each, independently, alkyl of 1-6 carbon atoms, alkenylof 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, hydroxyalkyl of 1-6carbon atoms, alkoxyalkyl of 2-12 carbon atoms, alkylthioalkyl of 2-12carbon atoms, alkylaminoalkyl of 2-12 carbon atoms, dialkylaminoalkyl of3-12 carbon atoms, arylalkyl of 7-10 carbon atoms, cycloalkyl of 3-8carbon atoms,--CF₃, --COR⁷, --CO₂ R⁷, --CONHR⁷, --SO₂ R⁷, or Ar;

A is --CH₂ --, --NR⁷ --, --O--, --S--, --SO--, --SO₂ --, --PR⁷ --,--CO--, --NHCO --, --NHSO--, or --P(O)(R⁷)--;

Ar is phenyl, naphthyl, pyridyl, quinolyl, isoquinolyl, quinoxalyl,thienyl, thionaphthyl, furyl, benzofuryl, benzodioxyl, benzoxazolyl,benzoisoxazolyl, indolyl, thiazolyl, isoxazolyl, pyrimidinyl, pyrazinyl,imidazolyl, benzopyranyl, benz[b]thiophenolyl, benzimidazolyl,benzthiazolyl, benzodioxolyl, piperidinyl, morpholinyl, piperazinyl,tetrahydrofuranyl, or pyrrolidinyl; wherein the Ar group may beoptionally mono-, di-, or tri- substituted with a group selected fromalkyl of 1-6 carbon atoms, arylalkyl of 7-10 carbon atoms, alkoxy of 1-6carbon atoms, cyano, halo, hydroxy, nitro, carbalkoxy of 2-7 carbonatoms, trifluoromethyl, amino, dialkylamino of 1-6 carbon atoms peralkyl group, dialkylaminoalkyl of 3-12 carbon atoms, hydroxyalkyl of 1-6carbon atoms, alkoxyalkyl of 2-12 carbon atoms, alkylthio of 1-6 carbonatoms, --SO₃ H, --PO₃ H, and --CO₂ H; ##STR6## is a nitrogen containingheterocycle that may be saturated, unsaturated, or partiallyunsaturated, and may be optionally mono-, di-, or tri- substituted witha group selected from alkyl of 1-6 carbon atoms, arylalkyl of 7-10carbon atoms, alkoxy of 1-6 carbon atoms, cyano, halo, hydroxy, nitro,carbalkoxy of 2-7 carbon atoms, trifluoromethyl, amino, dialkylamino of1-6 carbon atoms per alkyl group, dialkylaminoalkyl of 3-12 carbonatoms, hydroxyalkyl of 1-6 carbon atoms, alkoxyalkyl of 2-12 carbonatoms, alkylthio of 1-6 carbon atoms, --SO₃ H,--PO₃ H, and --CO₂ H;

with the proviso that R¹ and R² are not both hydrogen;

m=0-6;

n=0-6;

p=0-1;

q=0-1;

or a pharmaceutically acceptable salt thereof.

The pharmaceutically acceptable salts are those derived from suchinorganic cations such as sodium, potassium, and the like; organic basessuch as: mono-, di-, and trialkyl amines of 1-6 carbon atoms, per alkylgroup and mono-, di-, and trihydroxyalkyl amines of 1-6 carbon atoms peralkyl group, and the like; and organic and inorganic acids as: acetic,lactic, citric, tartaric, succinic, maleic, malonic, gluconic,hydrochloric, hydrobromic, phosphoric, nitric, sulfuric,methanesulfonic, and similarly known acceptable acids.

It is preferred that the aryl portion of the arylalkyl substituent is aphenyl, piperazinyl, piperidinyl, or pyridyl group that is optionallymono-, di-, or tri- substituted with a group selected from alkyl of 1-6carbon atoms, arylalkyl of 7-10 carbon atoms, alkoxy of 1-6 carbonatoms, cyano, halo, nitro, carbalkoxy of 2-7 carbon atoms,trifluoromethyl, amino, dialkylamino of 1-6 carbon atoms per alkylgroup, alkylthio of 1-6 carbon atoms, --SO₃ H, --PO₃ H, and --CO₂ H.

It is preferred that ##STR7## is a pyridyl, pyrazinyl, piperidinyl,morpholinyl, piperazinyl, pyrrolidinyl, thiazolyl, pyrimidinyl,isoxazolyl, pyrrolidinyl, or imidazolyl group that may be optionallysubstituted as described above.

Preferred members include those compounds in which R² is hydrogen; thosein which p=0 and B is Ar; those in which p=0, B is Ar, and R² ishydrogen; those in which p=0, B is Ar, R² is hydrogen, and Ar ispyridyl, furyl, piperazinyl, piperazinyl, morpholinyl, and piperidinyl;those in which m=0-3 and p=0; those in which m=2, n=0, p=1, q=1 and A is--O-- or NR⁷ ;

those in which R¹ is ##STR8## and R² is hydrogen; and those in which R¹is ##STR9## R² is hydrogen, and ##STR10## is an optionally substitutedmorpholinyl or piperazinyl group.

Of these compounds, preferred members also include those having thestructure ##STR11## wherein R¹ and R² are each, independently, hydrogen,or --CONH--[(CR³ R⁴)_(m) (--A--(CR⁵ R⁶)_(n))_(p) ]_(q) --B;

R³, R⁴, R⁵, R⁶, and B are each, independently, hydrogen, alkyl of 1-6carbon atoms, arylalkyl of 7-10 carbon atoms, cycloalkyl of 3-8 carbonatoms, --OR⁷, --SR⁷, halogen, --CN, --NO₂, --CF₃, --COR₇, --CONH₂, --SO₂R₇, --OSO₃ R⁷, --NR⁷ R⁸, --NHCOR⁷, --NHSO₂ R⁸, or Ar;

A is --CH₂ --, --NR⁷ --, --O--, --S--, --SO₂ --, --PR⁷ --, or--P(O)(R⁷)--;

R⁷ and R⁸ are each, independently, hydrogen, alkyl of 1-6 carbon atoms,arylalkyl of 7-10 carbon atoms, or Ar;

Ar is phenyl, naphthyl, pyridyl, quinolyl, isoquinolyl, quinoxalyl,thienyl, thionaphthyl, furyl, benzofuryl, benzodioxyl, benzoxazolyl,benzoisoxazolyl, or benzodioxolyl; wherein the Ar group may beoptionally mono-, di-, or tri- substituted with a group selected fromalkyl of 1-6 carbon atoms, arylallcyl of 7-10 carbon atoms, alkoxy of1-6 carbon atoms, cyano, halo, nitro, carbalkoxy of 2-7 carbon atoms,trifluoromethyl, amino, dialkylamino of 1-6 carbon atoms per alkylgroup, alkylthio of 1-6 carbon atoms, --SO₃ H, --PO₃ H, and --CO₂ H;

with the proviso that R¹ and R² are not both hydrogen;

m=0-6;

n=0-6;

p=0-1;

q=0-1;

or a pharmaceutically acceptable salt thereof.

The compounds of this invention carbamylated at the 42-position or atboth the 31- and 42-positions can be prepared by converting the 42-and/or 31-alcohols of rapamycin to a carbonate followed by reaction withan appropriately substituted amine to provide the desired carbamate.This scheme is illustrated below for the preparation of the compound ofExample 3. ##STR12##

Alternatively the compounds of this invention carbamylated at the42-position or at both the 31- and 42-positions can be prepared byreacting rapamycin with an appropriately substituted isocyanate underneutral conditions or in the presence of a base, such as pyridine.Preparation of carbamates of rapamycin using this method was disclosedin U.S. Pat. No. 5,118,678, which is hereby incorporated by reference.

The 31-carbamylated compounds of this invention can be prepared byprotecting the 42-alcohol of rapamycin with a protecting group, such aswith a tertbutyl dimethylsilyl group, followed by carbamylation of the31-position by the procedures described above. Removal of the protectinggroup provides the 31-carbamylated compounds. In the case of thetert-butyl dimethylsilyl protecting group, deprotection can beaccomplished under mildly acidic conditions.

Having the 31-position carbamylated and the 42-position deprotected, the42-position can be carbamylated using a different amine (via thecarbonate) or isocyanate than was reacted with the 31-alcohol, to givecompounds having different carbamates at the 31- and 42-positions.Alternatively, the 42-carbamylated compounds, prepared as describedabove, can be reacted with a different amine (via the carbonate) orisocyanate to provide compounds having different carbamates at the 31-and 42-positions.

The amines and isocyanates used to prepare the compounds of theinvention are commercially available or can be prepared by methods thatare disclosed in the literature.

This invention also covers analogous carbamates of other rapamycins suchas, but not limited to, 29-demethoxyrapamycin, [U.S. Pat. No. 4,375,464,32-demethoxyrapamycin under C.A. nomenclature]; rapamycin derivatives inwhich the double bonds in the 1--, 3--, and/or 5-positions have beenreduced [U.S. Pat. No. 5,023,262]; 42-oxorapamycin [U.S. Pat. No.5,023,262]; 29-desmethylrapamycin [U.S. Pat. No. 5,093,339,32-desmethylrapamycin under C.A. nomenclature];7,29-bisdesmethylrapamycin [U.S. Pat. No. 5,093,338,7,32-desmethylrapamycin under C.A. nomenclature]; and 15-hydroxy- and15,27-bishydroxy rapamycin [U.S. Pat. No. 5,102,876]. The disclosures inthe above cited U.S. Patents are hereby incorporated by reference.

Immunosuppressive activity for representative compounds of thisinvention was evaluated in an in vitro standard pharmacological testprocedure to measure lymphocyte proliferation (LAF) and in an in vivostandard pharmacological test procedure which evaluated the survivaltime of a pinch skin graft.

The comitogen-induced thymocyte proliferation procedure (LAF) was usedas an in vitro measure of the immunosuppressive effects ofrepresentative compounds. Briefly cells from the thymus of normal BALB/cmice are cultured for 72 hours with PHA and IL-1 and pulsed withtritiated thymidine during the last six hours. Cells are cultured withand without various concentrations of rapamycin, cyclosporin A, or testcompound. Cells are harvested and incorporated radioactivity isdetermined. Inhibition of lymphoproliferation is assessed as percentchange in counts per minute from nondrug treated controls. For eachcompound evaluated, rapamycin was also evaluated for the purpose ofcomparison. An IC₅₀ was obtained for each test compound as well as forrapamycin. When evaluated as a comparator for the representativecompounds of this invention, rapamycin had an IC₅₀ ranging from 0.3-4.6nM. The results obtained for the representative compounds of thisinvention were also expressed as a ratio compared with rapamycin. Apositive ratio indicates immunosuppressive activity. A ratio of greaterthan 1 indicates that the test compound inhibited thymocyteproliferation to a greater extent than rapamycin. Calculation of theratio is shown below. ##EQU1##

Representative compounds of this invention were also evaluated in an invivo test procedure designed to determine the survival time of pinchskin graft from male BALB/c donors transplanted to male C₃ H(H-2K)recipients. The method is adapted from Billingham R. E. and Medawar P.B., J. Exp. Biol. 28:385-402, (1951). Briefly, a pinch skin graft fromthe donor was grafted on the dorsum of the recipient as an allograft,and an isograft was used as control in the same region. The recipientswere treated with varying concentrations of either test compoundsintraperitoneally or orally. Rapamycin was used as a test control.Untreated recipients served as rejection control. The graft wasmonitored daily and observations were recorded until the graft becamedry and formed a blackened scab. This was considered as the rejectionday. The mean graft survival time (number of days±S.D.) of the drugtreatment group was compared with the control group. The following tableshows the results that were obtained. Results are expressed as the meansurvival time in days. Untreated (control) pinch skin grafts are usuallyrejected within 6-7 days. The results shown in Table 1 are based on adose of 4 mg/kg of test compound. A survival time of 12.0±1.7 days wasobtained for rapamycin at 4 mg/kg.

The following table summarizes the results of representative compoundsof this invention in these two standard test procedures.

                  TABLE 1                                                         ______________________________________                                        EVALUATION OF IMMUNOSUPPRESSIVE ACTIVITY*                                              LAF           Skin Graft                                             Compound  IC.sub.50 (nM)                                                                             (ratio) (days ± SD)                                 ______________________________________                                        Example 2 3.4          1.4     11.2 ± 1.0                                            0.4          1.1     10.0 ± 0.9                                  Example 3 0.2          6.2      9.7 ± 0.8                                  Example 4 1.2          1.2     10.0 ± 0.0                                            0.6          1.0                                                              1.2          0.5                                                    Example 5 1.5          0.6     11.2 ± 1.2                                  Example 6 4.1          1.1     12.0 ± 1.0                                  Example 7 1.1          1.5      9.3 ± 0.8                                            0.9          1.0                                                    Example 8 6.5          0.3                                                    Example 9 0.2          1.1      8.7 ± 0.5                                  Example 11                                                                              1.9          0.26    12.6 ± 1.5                                            2.2          0.4     12.0 ± 0.6                                            1.9          0.5                                                    Example 12                                                                              2.8          0.4     12.4 ± 0.9                                            3.0          0.9     11.0 ± 1.7                                  Example 13                                                                              1.0          0.5     11.2 ± 0.7                                            1.9          0.4     11.7 ± 1.6                                  Example 14                                                                              1.2          0.6     13.6 ± 0.6                                            2.2          0.4                                                              3.0          0.8                                                              1.4          0.5                                                    Example 15                                                                              3.5          0.7                                                    Example 16                                                                              4.0          0.6                                                    Example 17                                                                              0.8          0.9     11.8 ± 1.5                                  Example 18                                                                              1.6          0.4     13.0 ±  0.7                                           1.0          0.7                                                    Example 21                                                                              0.9          0.6     11.0 ± 1.1                                            1.4          0.8                                                              1.6          0.6                                                    Example 22                                                                              0.5          0.9     10.8 ± 1.2                                            1.9          0.3                                                    Example 23                                                                              0.9          0.4      9.2 ± 1.5                                  Example 24                                                                              1.8          0.5      8.7 ± 1.0                                  Example 25                                                                              0.5          1.8     11.7 ± 0.8                                            2.6          0.9                                                              1.9          0.8                                                    Example 26                                                                              0.9          1.1     12.5 ± 0.6                                            0.7          0.9                                                    Example 27                                                                              1.9          1.3                                                    Example 28                                                                              2.3          1.0                                                    Example 29                                                                              1.6          0.9                                                    Example 30                                                                              1.7          0.9                                                    Example 32                                                                              2.6          0.6                                                    Example 33                                                                              1.6          0.9                                                    Example 36                                                                              0.7          1.3     11.3 ± 0.8                                  Example 37                                                                              5.1          0.6     13.0 ± 0.6                                                                 10.2 ± 1.2                                  Example 38                                                                              2.4          0.5     11.3 ± 1.0                                  Example 39                                                                              2.6          0.4     10.8 ± 1.2                                  Example 40                                                                              4.8          0.2     11.2 ± 1.3                                  Example 41                                                                              18.0         0.1     11.3 ± 1.6                                  Example 42                                                                              0.5          1.6                                                              2.2          1.1                                                    Example 43                                                                              0.3          1.7      9.0 ± 0.0                                  Example 44                                                                              0.7          0.7                                                    Example 45                                                                              0.5          0.8      8.3 ± 0.5                                  Example 46                                                                              2.0          0.4      7.5 ± 0.5                                  Example 47                                                                              12.0         0.1      7.5 ± 0.8                                  ______________________________________                                         *Calculation of the ratio was described supra.                           

The results of these standard pharmacological test proceduresdemonstrate immunosuppressive activity both in vitro and in vivo for thecompounds of this invention. Positive ratios in the LAF test proceduresindicates suppression of T-cell proliferation, thereby demonstrating theimmunosuppressive activity of the compounds of this invention. Astransplanted pinch skin grafts are typically rejected within 6-7 dayswithout the use of an immunosuppressive agent, the increased survivaltime of the skin graft when treated with the compounds of this inventionfurther demonstrates their utility as immunosuppressive agents and asagents useful in the treatment or prevention of transplantationrejection.

Representative compounds of this invention were also evaluated in theadjuvant arthritis standard pharmacological test procedure, whichmeasures the ability of the compound tested to inhibit immune mediatedinflammation. The adjuvant arthritis test procedure is a standardpharmacological test procedure for rheumatoid arthritis. The procedureused and results obtained are described below

The adjuvant arthritis standard pharmacological test procedure measuresthe ability of test compounds to prevent immune mediated inflammationand inhibit or treat rheumatoid arthritis. The following brieflydescribes the test procedure used. A group of rats (male inbread WistarLewis rats) are pre-treated with the compound to be tested (1 h prior toantigen) and then injected with Freud's Complete Adjuvant (FCA) in thefight hind paw to induce arthritis. The rats are then orally dosed on aMonday, Wednesday, Friday schedule from day 0-14 for a total of 7 doses.Both hind paws are measured on days 16, 23, and 30. The difference inpaw volume (mL) from day 16 to day 0 is determined and a percent changefrom control is obtained. The left hind paw (uninjected paw)inflammation is caused by T-cell mediated inflammation and is recordedin the above table (% change from control). The fight hind pawinflammation, on the other hand, is caused by nonspecific inflammation.Compounds were tested at a dose of 2 mg/kg. The results are expressed asthe percent change in the uninjected paw at day 16 versus control; themore negative the percent change, the more potent the compound.Rapamycin provided -70-87% change versus control, indicating thatrapamycin treated rats had 70 to 87% less immune induced inflammationthan control rats. The following results were obtained: Example 6, -38%change versus control; Example 14, -38 and -21% change versus control;Example 17, -30% change versus control; Example 18, -14% versus control;Example 26, -70% change versus control; and Example 13 showed no reducedinflammation at 2 mg/kg.

Based on the results of these standard pharmacological test procedures,the compounds are useful in the treatment or prevention oftransplantation rejection such as kidney, heart, liver, lung, bonemarrow, pancreas (islet cells), cornea, small bowel, and skinallografts, and heart valve xenografts; in the treatment of autoimmunediseases such as lupus, rheumatoid arthritis, diabetes mellitus,myasthenia gravis, and multiple sclerosis; and diseases of inflammationsuch as psoriasis, dermatitis, eczema, seborrhea, inflammatory boweldisease, pulmonary inflammation, asthma, and eye uveitis.

Because of the activity profile obtained, the compounds of thisinvention also are considered to have antitumor, antifungal activities,and antiproliferative activities. The compounds of this inventiontherefore are also useful in treating solid tumors, fungal infections,and hyperproliferative vascular diseases such as restenosis andatherosclerosis.

It is contemplated that when the compounds of this invention are used asan immunosuppressive or antiinflammatory agent, they can be administeredin conjunction with one or more other immunoregulatory agents. Suchother immunoregulatory agents include, but are not limited toazathioprine, corticosteroids, such as prednisone andmethylprednisolone, cyclophosphamide, rapamycin, cyclosporin A, FK-506,OKT-3, and ATG. By combining the compounds of this invention with suchother drugs or agents for inducing immunosuppression or treatinginflammatory conditions, lesser amounts of each of the agents arerequired to achieve the desired effect. The basis for such combinationtherapy was established by Stepkowski whose results showed that the useof a combination of rapamycin and cyclosporin A at subtherapeutic dosessignificantly prolonged heart allograft survival time. [TransplantationProc. 23: 507 (1991)].

The compounds of this invention can be formulated neat or with apharmaceutical carrier to a mammal in need thereof. The pharmaceuticalcarrier may be solid or liquid.

A solid carrier can include one or more substances which may also act asflavoring agents, lubricants, solubilizers, suspending agents, fillers,gildants, compression aids, binders or tablet-disintegrating agents; itcan also be an encapsulating material. In powders, the carrier is afinely divided solid which is in admixture with the finely dividedactive ingredient. In tablets, the active ingredient is mixed with acarrier having the necessary compression properties in suitableproportions and compacted in the shape and size desired. The powders andtablets preferably contain up to 99% of the active ingredient. Suitablesolid carriers include, for example, calcium phosphate, magnesiumstearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose,methyl cellulose, sodium carboxymethyl cellulose, polyvinylpyrrolidine,low melting waxes and ion exchange resins.

Liquid carriers are used in preparing solutions, suspensions, emulsions,syrups, elixirs and pressurized compositions. The active ingredient canbe dissolved or suspended in a pharmaceutically acceptable liquidcarrier such as water, an organic solvent, a mixture of both orpharmaceutically acceptable oils or fats. The liquid carrier can containother suitable pharmaceutical additives such as solubilizers,emulsifiers, buffers, preservatives, sweeteners, flavoring agents,suspending agents, thickening agents, colors, viscosity regulators,stabilizers or osmo-regulators. Suitable examples of liquid carriers fororal and parenteral administration include water (partially containingadditives as above, e.g. cellulose derivatives, preferably sodiumcarboxymethyl cellulose solution), alcohols (including monohydricalcohols and polyhydric alcohols, e.g. glycols) and their derivatives,and oils (e.g. fractionated coconut oil and arachis oil). For parenteraladministration, the carrier can also be an oily ester such as ethyloleate and isopropyl myristate. Sterile liquid carriers are useful insterile liquid form compositions for parenteral administration. Theliquid carrier for pressurized compositions can be halogenatedhydrocarbon or other pharmaceutically acceptable propellant.

Liquid pharmaceutical compositions which are sterile solutions orsuspensions can be utilized by, for example, intramuscular,intraperitoneal or subcutaneous injection. Sterile solutions can also beadministered intravenously. The compound can also be administered orallyeither in liquid or solid composition form.

The compounds of this invention may be administered rectally in the formof a conventional suppository. For administration by intranasal orintrabronchial inhalation or insuffiation, the compounds of thisinvention may be formulated into an aqueous or partially aqueoussolution, which can then be utilized in the form of an aerosol. Thecompounds of this invention may also be administered transdermallythrough the use of a transdermal patch containing the active compoundand a carrier that is inert to the active compound, is non toxic to theskin, and allows delivery of the agent for systemic absorption into theblood stream via the skin. The carrier may take any number of forms suchas creams and ointments, pastes, gels, and occlusive devices. The creamsand ointments may be viscous liquid or semisolid emulsions of either theoil-in-water or water-in-oil type. Pastes comprised of absorptivepowders dispersed in petroleum or hydrophilic petroleum containing theactive ingredient may also be suitable. A variety of occlusive devicesmay be used to release the active ingredient into the blood stream suchas a semipermiable membrane covering a reservoir containing the activeingredient with or without a carrier, or a matrix containing the activeingredient. Other occlusive devices are known in the literature.

In addition, the compounds of this invention may be employed as asolution, cream, or lotion by formulation with pharmaceuticallyacceptable vehicles containing 0.1-5 percent, preferably 2%, of activecompound which may be administered to a fungally affected area.

The dosage requirements vary with the particular compositions employed,the route of administration, the severity of the symptoms presented andthe particular subject being treated. Based on the results obtained inthe standard pharmacological test procedures, projected daily dosages ofactive compound would be 0.1 μg/kg-100 mg/kg, preferably between0.001-25 mg/kg, and more preferably between 0.01-5 mg/kg. Treatment willgenerally be initiated with small dosages less than the optimum dose ofthe compound. Thereafter the dosage is increased until the optimumeffect under the circumstances is reached; precise dosages for oral,parenteral, nasal, or intrabronchial administration will be determinedby the administering physician based on experience with the individualsubject treated. Preferably, the pharmaceutical composition is in unitdosage form, e.g. as tablets or capsules. In such form, the compositionis sub-divided in unit dose containing appropriate quantifies of theactive ingredient; the unit dosage forms can be packaged compositions,for example, packeted powders, vials, ampoules, prefilled syringes orsachets containing liquids. The unit dosage form can be, for example, acapsule or tablet itself, or it can be the appropriate number of anysuch compositions in package form.

The following examples illustrate the preparation of representativecompounds of this invention.

Example 1

Rapamycin 42-nitrophenyl carbonate

A solution of 2.0 g of rapamycin in 10 ml of dichloromethane and 2 mL ofdry pyridine was cooled to -78° C. under a nitrogen atmosphere. To thissolution, 662 mg 4-nitrophenyl chloroformate was added; the resultingsolution was stirred at room temperature under nitrogen for 20 hours.The mixture was diluted with water and extracted with dichloromethane.The dichloromethane extract was washed with water, dried over MgSO₄ andevaporated. The residue was chromatographed on silica gel. Elution with33% ethyl acetate in n-hexane gave 2.07 g of rapamycin 42-p-nitrophenylcarbonate as a white foam.

Example 2

Rapamycin 42-ester with 4-(3-hydroxypropyl)piperazine-1-carboxylic acid

A solution of 130 mg (0.90 mmole) of 1-(3-hydroxypropyl)piperazine in 2mL dichloromethane was added to a solution of 320 mg (0.30 mmole)rapamycin-42-(4-nitrophenyl)carbonate in 6 mL dichloromethane undernitrogen at -5° C. and allowed to warm to 20° with stirring. After 4hours, the reaction mixture was partitioned between dichloromethane andwater/brine. The organic portion was washed with brine and flashchromatographed through silica gel using methanol (2.0 to 3.0%) indichloromethane, yielding 115 mg product as a white solid, mp 104°-113°C.

IR (KBr): 3430, 2930, 1715, 1640,1450, 1240, and 985 cm⁻¹.

NMR (CDCl3, 400 MHz): δ3.81(t (J=5.2 Hz), 2H, Hd); 3.49 (broad, 4H, Ha);3.38 (s, 3H, OMe); 3.33 (s, 3H, OMe); 3.13 (s, 3H, OMe); 2.62 (t (J=5.4Hz), 2H, Hc); 2.48 (broad, 4H, Hb) ppm.

MS (neg ion FAB): 1083 (M⁻), 590. ##STR13##

Example 3

Rapamycin 42-ester with morpholine-4-carboxylic acid

A solution of 95 mg (1.1 mmole) morpholine in 1 mL dry dichloromethanewas added to a stirred solution of 330 mg (0.31 mmole)rapamycin-42-(4-nitrophenyl)carbonate in 6 mL dichloromethane at -5° C.under nitrogen; stirring was continued 4.5 hours at -5° and 2 hours at20°. The reaction mixture was partitioned between dichloromethane andwater/brine; the organic portion was washed with brine and flashchromatographed through silica gel using methanol (1.0 to 1.6%) indichloromethane, yielding 70 mg product as a white solid, mp 105°-115°C.

IR (KBr): 3450, 2950, 1710, 1650, 1250, and 993 cm⁻¹.

NMR (CDCl3, 400MHz): δ3.64 (4H, 3-morpholine); 3.46 (t (J=4.9 Hz), 4H,2-morpholine); 3.37 (s, 3H, OMe); 3.32 (s, 3H, OMe); 3.12 (s, 3H, OMe)ppm.

MS (neg ion FAB): 1026 (M⁻), 590.

Example 4

Rapamycin 42-ester with 4-methylpiperazine-1-carboxylic acid

A solution of 95 mg (0.95 mmole) 1-methylpiperazine in 2 mLdichloromethane was added to a solution of 310 mg (0.29 mmole)rapamycin-42-(4-nitrophenyl)carbonate in 6 mL dichloromethane at 0° C.under nitrogen and stirred at 0° for 2 hours and at 20° for 2 hours. Thereaction mixture was partitioned between dichloromethane andwater/brine. The organic portion was washed with brine and flashchromatographed through silica gel using methanol (2.0 to 3.0%) indichloromethane, yielding 120 mg product as a white solid, mp 108°-116°C.

IR (KBr): 3450, 2945, 1710, 1650, 1460, 1240, 1110, and 990 cm⁻¹.

NMR (CDCl3, 400 MHz): δ3.50 (broad, 4H, 2-piperazine); 3.39 (s, 3H,OMe); 3.33 (s, 3H, OMe); 3.14 (s, 3H, OMe); 2.36 (broad, 4H,3-piperazine); 2.30 (s, 3H, NMe) ppm.

MS (neg ion FAB): 1039 (M⁻), 590.

Example 5

Rapamycin 42-ester with 4-methyl-piperazine-1-carboxylic acid salt withhydrochloric acid

A solution of 720 mg rapamycin 42-ester with4-methyl-piperazine-1-carboxylic acid in a mixture of 5 ml ethyl acetateand 10 ml ether was treated at 0° under nitrogen with 0.7 ml 1M HCl(gas) in ether. The hydrochloride salt formed instantly. Stirring wascontinued at 0° under nitrogen for 1/4 hour. The solid product wascollected by filtration, washed with ether, and dried in vacuum at 55°to afford 510 mg of the title product as a white solid, mp 128°-134°.

IR (KBr): 3400 (OH, NH), 1715 (lactone and ketone C═O). 1640 (amideC═O), 1450, 1260, 1100, and 980 cm⁻¹.

¹ HNMR (DMSO-D₆, 400 MHz δ4.39 (m, 1H, C₄₂ -H), 3.29, 3.14, 3.04 (eachs, 3H, OCH₃), 2.74 (s, 3H, CH₃ --N--) ppm.

MS (neg. ion FAB): 1039.6(M⁻), 590.4, 447.4. C/H/N analysis for C₅₇ H₈₉N₃ O14.HCl.H₂ O; Calc. 62.53/8.28/3.83; Found: 62.42/8.48/3.77.

Example 6

Rapamycin 42-ester with piperazine-1-carboxylic acid

A solution of 190 mg (2.2 mmole) piperazine in 4 mL dichloromethane wasadded to a solution of 550 mg (0.51 mmole)rapamycin-42-(4-nitrophenyl)carbonate in 12 mL dichloromethane at 0° C.under nitrogen and stirred 45 minutes. Partitioning betweendichloromethane and water/brine, washing with brine and flashchromatography through silica gel using 5% methanol in dichloromethaneyielded 350 mg product as a pale yellow solid, mp 120°-131° C.

IR (KBr): 3460, 2950, 1705, 1650, 1460, 1245, and 990 cm⁻¹.

NMR (CDCl3, 400 MHz): δ4.8 (broad, 1H, NH); 3.46 (broad, 4H,2piperazine); 3.39 (s, 3H, OMe); 3.33 (s, 3H, OMe); 3.14 (s, 3H, OMe);2.83 (broad, 4H, 3-piperazine) ppm.

MS (neg ion FAB): 1025 (M⁻), 590.

Example 7

Rapamycin 42-ester with 4-(2-hydroxyethyl)piperazine-1-carboxylic acid

A solution of 1-(2-hydroxyethyl)piperazine) (130 rag, 1.0 mmole) in 1 mLdry dichloromethane was added to a solution of 330 mg rapamycin42-p-nitrophenyl)carbonate (0.31 mmole) in 6 mL dry dichloromethane at-8° under nitrogen and stirred at -8° for 1.5 hours. The reactionmixture was partitioned between dichloromethane and water/brine, theaqueous portion was extracted with dichloromethane, the combined organicportion was washed with brine, dried over MgSO₄ and evaporated to awhite solid foam. Flash chromatography through silica gel using 2%methanol in dichloromethane yielded 140 mg of the title compound as awhim solid, mp 112°-120° C.

IR (KBr): 3450, 2950, 1725, 1650, 1460, 1250 and 995 cm⁻¹.

NMR (CDCl3, 400 MHz): δ3.64 (t (J=5.2Hz), 2H, Hd); 3.51 (broad, 4H, Ha);3.39 (s, 3H, OMe); 3.33 (s, 3H, OMe); 3.14 (s, 3H, OMe); 2.57 (t (J=5.2Hz), 2H, Hc); 2.49 (broad, 4H, Hb) ppm.

MS (neg. ion FAB): m/z at 1069 (m⁻), 590. ##STR14##

Example 8

Rapamycin 42-ester with 2-(dimethylamino)ethylcarbamic acid salt withhydrochloric acid

A solution of 100 mg rapamycin 42-p-nitrophenyl carbonate in 5 mldichloromethane was treated at 0° under nitrogen with 44 mgN,N-dimethylethylenediamine in 0.5 ml dichloromethane. The reactionmixture was stirred at 0° under nitrogen for 1/2 hour, diluted with 50ml dichloromethane, washed with water, and dried with MgSO₄. The solventwas evaporated and the residue was dissolved in 1.5 ml dichloromethane.The dichloromethane solution was cooled to 0° under nitrogen and wastreated with 1.5 ml 0.1 M HCl (gas) in ether. The resulting solution wasdiluted with 10 ml ether and the hydrochloride salt precipitated out.Stirring was continued at 0° under nitrogen for 1/4 hour. The productwas collected by filtration, washed with ether, and dried at 54° invacuum to afford 80 mg of the title product as a white solid, mp125°-130°.

IR(KBr): 3400 (OH and NH), 1715 (lactone and ketone C═O), 1640 (amideC═O), 1450, 1240, 1090 and 985 cm⁻¹.

¹ HNMR (DMSO-D₆, 400 MHz): δ9.85 (s, 1H, NH⁺), 7.35 (s, 1H,--OC(O)NH--), 4.40 (m, 1H, N(CH₃)₂), 3.26, 3.15, 3.04 (each s, 3H,OCH₃), 2.76 (s, 6H, N(CH₃)₂) ppm.

MS (neg. ion FAB): 1027.6 (M⁻), 590.4, 435.3. C/H/N analysis for C₅₆ H₈₉N₃ O₁₄.HCl.2H₂ O; Calc. 61.09/8.60/3.81; Found 61.06/8.55/3.91.

Example 9

Rapamycin 42-ester with [(4-aminophenyl)methyl]carbamic acid

A solution of 250 mg rapamycin 42-p-nitrophenyl carbonate in 13 mldichloromethane was treated at -10° under nitrogen with 145 mg4-aminobenzylamine in 0.5 ml dichloromethane. The reaction mixture wasstirred at room temperature under nitrogen for 2 hours, diluted with 150ml dichloromethane, washed with water and dried with MgSO₄. The solventwas evaporated and the residue chromatoghraphed on silica gel. Elutionwith methanol/dichloromethane (2/98) afforded 140 mg of the titleproduct as a white solid, mp 107°-110°.

IR (KBr): 3400 (OH and NH), 1720 (lactone and ketone C═O), 1635 (amideC═O), 1520, 1450, 1240, 1100,985, and 750 cm⁻¹.

¹ HNMR (CDCl₃,400MH_(z)): δ7.08(d, J=10 cps, 2H, protons a), 6.65 (d,J=10 cps, 2H, protons b), 4.60 (m, 1H, C₄₂ -H) 4.24 (d, 2H benzylicprotons), 3.39, 3.33, 3.14 (each s, 3H, OCH₃) ppm.

MS (neg. ion FAB): 1061.6 (M⁻), 590.4, 469.3. ##STR15##

Example 10

Rapamycin 42-ester with [(4-aminophenyl)methyl]carbamic acid salt withhydrochloric acid

A solution of 500 mg rapamycin 42-ester with [(4-aminophenyl)methyl]carbamic acid in a mixture of 2 ml ethyl acetate and 8 ml ether wastreated at -10° under nitrogen with 0:5 ml 1M HCl (gas) in ether. Thehydrochloride salt formed instantly. Stirring was continued at -10°under nitrogen for 1/4 hour. The product was collected by filtration,washed with ether, and dried in vacuum to afford 460 mg of the titleproduct as a yellow solid, mp 110°-115° (dec.).

IR (KBr): 3400 (OH and NH), 1715 (lactone and ketone C═O), 1640 (amideC═O), 1505, 1440, 1140, 1090, and 985 cm⁻¹.

¹ HNMR (DMSO-D₆, 400 MHz): δ7.29 (d, J=10 cps, 2H, protons b), 7.24 (d,J-10 cps, 2H, protons a), 4.40 (m, 1H, C₄₂ -H), 3.26, 3.15, 3.04 (eachs, 3H, OCH₃) ppm.

MS (neg. ion FAB): 1061.1 (m⁻), 590.1,469.1. ##STR16##

Example 11

Rapamycin 42-ester with (3-dimethylaminopropyl)carbamic acid

A solution of 16.0 g rapamycin 42-p-nitrophenyl carbonate in 250 mldichloromethane was treated at -10° under nitrogen with 2.96 g3-dimethylaminopropylamine in 50 ml dichloromethane. The reactionmixture was stirred at 0° under nitrogen for 1.0 hour, diluted with 900ml dichloromethane, washed with water and dried with MgSO₄. The solventwas evaporated and the residue chromatographed on silica gel. Elutionwith methanol]ethyl acetate (1/3) afforded 7.1 g of the title product asa white solid, mp 104°-106°.

IR (KBr): 3400 (OH and NH), 1715 (lactone and ketone C═O), 1640 (amideC═O), 1440, 1365, 1250, 1100, and 985 cm⁻¹.

¹ HNMR (CDCl₃, 400 MHz): δ4.55 (m, 1H, C₄₂ -H), 3.39, 3.33, 3.14 (eachs, 3H, OCH₃), 3.25 (t, 2H, --CH₂ NHC(O)--), 2.35 (t, 2H, ##STR17## 2.22(s, 6H, (CH₃)₂ N) ppm.

MS (neg. ion FAB): 1041.6 (M⁻), 590.4, 449.4. C/H/N analysis for C₅₇ H₉₁N₃ O₁₄.H₂ O; Calc: 64.56/8.84/3.94 Found: 64.53/8.80/3.95.

Example 12

Rapamycin 42-ester with (3-dimethylaminopropyl)carbamic acid salt withhydrochloric acid

A solution of 520 mg rapamycin 42-ester with(3-dimethylaminopropyl)carbamic acid in a mixture of 4 ml ethyl acetateand 12 ml ether was treated at 0° under nitrogen with 0.75 ml 1M HCl(gas) in ether. The hydrochloride salt formed instantly. Stirring wascontinued at 0° under nitrogen for 1/4 hour. The product was collectedby filtration, washed with ether and dried in vacuum to give 460 mg ofthe title product as a pale solid, mp 108°-112° (dec.). IR (KBr): 3400(OH and NH), 1716 (lactone and ketone C═O), 1640 (amide C═O), 1455,1240, 1100 and 985 cm⁻¹.

¹ HNMR (DMSO-D₆, 400 MHz): δ4.40 (m, 1H, C₄₂ -H), 3.27, 3.15, 3.04 (eachs, 3H, OCH₃), 2.72 (s, 6H, --N(CH₃)₂) ppm.

MS (neg. ion FAB); 1041.6 (m⁻), 590.4, 449.4. C/H/N analysis for C₅₇ H₉₁N₃ O₁₄.HCl.H₂ O; Calc.: 62.41/8.54/3.83. Found: 62.33/8.66/3.71.

Example 13

Rapamycin 42-ester with (2-diethylamino-ethyl) carbamic acid

A solution of 480 mg rapamycin 42-p-nitrophenyl carbonate in 8 mldichloromethane was treated at 0° under nitrogen with 136 mgN,N-diethylethylenediamine in 1 ml dichloromethane. The reaction mixturewas stirred at 0° under nitrogen for 2 hours, diluted with 200 mldichloromethane, washed with water and dried with MgSO₄. The solvent wasevaporated and the residue chromatographed on silica gel. Elution withmethanol/dichloromethane (5/95) afforded 405 mg of the title product asa white solid, mp 101°-104°.

IR (KBr): 3400 (OH and NH), 1720 (lactone and ketone C═O), 1650 (amideC═O), 1460, 1350, 110 and 990 cm⁻¹.

¹ HNMR (CDCl3, 400 MHz): a 4.54 (m, 1H, C₄₂ -H), 3.37, 3.31, 3.15 (eachs, 3H, OCH₃), 2.55 (m, 6H, protons a) 2.70 (m, 2H, protons b) ppm.##STR18## MS (neg. ion FAB): 1055.4 (M⁻), 590.2, 463.2.

Example 14

Rapamycin 42-ester with (2-diethylamino-ethyl) carbamic acid salt withhdyrochloric acid

A solution of 270 mg rapamycin 42-ester with (2-diethylamino-ethyl)carbamic acid in a mixture of 2.5 ml ethyl acetate and 8 ml ether wastreated at 0° under nitrogen with 0.26 ml 1M HCl (gas) in ether. Thehydrochloride salt formed instantly. Stirring was continued at 0° undernitrogen for 1/4 hour. The product was collected by filtration, washedwith ether, and dried in vacuum at 50° to afford 170 mg of the titleproduct as a pale solid, mp 101°-106°.

IR (KBr): 3400 (OH and NH), 1720 (lactone and ketone C═O), 1650 (amideC═O), 1450, 1400, 1250, 1100 and 990 cm⁻¹.

¹ HNMR (DMSO-D₆, 400 MHz): δ4.39 (m, 1H, C₄₂ -H), 3.25, 3.15, 3.04 (eachs, 3H, OCH₃), 3.05-3.13 (m, 8H, --CH₂ --N--), 1.19 (t, 6H, --N--C--CH₃)ppm.

MS (neg. ion FAB): 1055.7 (M⁻), 590.3, 463.3.

Example 15

Rapamycin 42 -ester with (2-diethylamino-ethyl) carbamic acid salt withmaleaic acid

A solution of 540 mg rapamycin 42-ester with (2-diethylamino-ethyl)carbamic acid in a mixture of 5 ml ethyl acetate and 5 ml ether wastreated at room temperature under nitrogen with 60 mg maleic acid in amixture of 1 ml ethyl acetate and 1 ml ether. The resulting mixturebecame cloudy and turned into a suspension, which was warmed to 50° toget a solution. The clear solution was evaporated at 50° under reducedpressure and dried in vacuum to afford 560 mg of the title product as awhite solid, mp 101°-105°.

IR (KBr): 3410 (OH and NH), 1720 (lactone, ketone and acid C═O), 1650(amide C═O), 1460, 1400, 1350, 1250, 1100, and 995 cm⁻¹.

¹ HNMR (DMSO-D₆): δ6.03 (s, 2H, maleate olefin), 4.40 (m, 1H, C₄₂ -H),3.27, 3.16, 3.05 (each s, 3H, OCH₃), 3.26-3.16 (m, 8H, --CH₂ --N), 1.18(t, 6H, CH₃ --C--N<) ppm.

MS (neg. ion FAB): 1055.7 (M⁻), 590.4, 463.0.

Example 16

Rapamycin 42-ester with (2-diethylamino-ethyl) carbamic acid salt withcitric acid

A solution of 528 mg rapamycin 42-ester with(2-diethylamino-ethyl)carbamic acid in a mixture of 2 ml ethyl acetateand 8 ml ether was treated at room temperature with 96 mg citric acid in1 ml methanol. The resulting solution was stirred at room temperatureunder nitrogen for 1/4 hour. The solvent was evaporated and the residuewas dried in vacuum at 50° to afford 590 mg of the title product as awhite solid, mp 96°-100°.

IR (KBr): 3430 (OH and NH), 1720 (lactone, ketone and acid C═O), 1640(amide C═O), 1440, 1250, 1190, 1090 and 995 cm⁻¹.

¹ HNMR (DMSO-D₆): δ7.24 (m, 1H, --NHCO), 4.38 (m, 1H, C₄₂ -H), 3.25,3.14, 3.08 (each s, 3H, OCH₃), 1.11 (t, 6H, --N--C--CH₃) ppm.

MS (neg. ion FAB): 1055.6 (m⁻), 590.3, 463.0.

Example 17

Rapamycin 42-ester with 4(1-ethyl-pyrrolidin-2-ylmethyl) carbamic acid

A solution of 480 mg rapamycin 42-p-nitrophenyl carbonate in 5 mldichloromethane was treated at -10° under nitrogen with 128.2 mg2-(aminomethyl)-1-ethylpyrrolidine in 1 ml dichloromethane. The reactionmixture was stirred at 0°/N₂ for 4 hours and at room temperature/N₂ for1/2 hour, diluted with 150 ml dichloromethane, washed with water, anddried with MgSO₄. The solvent was evaporated and the residuechromatographed on silica gel. Elution with methanol/dichloromethane(5/95) afforded 171 mg of the title product as pale yellow solid, mp115°-118°.

IR (KBr): 3400 (OH and NH), 1715 (lactone and ketone C═O), 1640 (amideC═O), 1450, 1250, 1100, and 990 cm⁻¹.

¹ HNMR (CDCl3, 400 MHz): δ4.55 (m, 1H, C₄₂ -H), 3.36, 3.32, 3.12 (eachs, 3H, OCH₃) ppm.

MS (neg. ion FAB): 1067.2 (M⁻), 590.1,475.1.

Example 18

Rapamycin 42-ester with 4-(1-ethyl-pyrrolidin-2-ylmethyl-carbamic acidsalt with hydrochloric acid

A solution of 520 mg rapamycin 42-ether, with4-(1-ethyl-pyrrolidin-2-ylmethyl) carbamic acid in a mixture of 6 mlethyl acetate and 10 ml dry ether was treated at 0° under nitrogen with0.5/ml 1M HCl (gas) in ether. The hydrochloride salt formed instantly.Stirring was continued at 0° under nitrogen for 1/2 hour. The productwas collected by filtration, washed with ether and dried in vacuum at54° for 16 hours to give 360 mg of the title product as a pale solid, mp113°-120° (dec).

IR (KBr): 3400 (OH and NH), 1720 (lactone and ketone C═O), 1650 (amideC═O), 1455, 1250, 1100, and 990 cm⁻¹.

¹ HNMR (DMSO-D₆, 400 MHz): δ4.40 (m, 1H, C₄₂ -H), 3.27, 3.15, 3.05 (eachs, 3H, OCH₃) ppm.

MS (neg. ion FAB): 1067.2 (M⁻), 590.1, 475.1. C/H/N/analysis for C₅₉ H₉₃N₃ O₁₄.HCl.H₂ O; Calc.: 63.10/8.61/3.7; Found; 63.01/8.58/3.63.

Example 19

Rapamycin 42-ester with piperidine-1-carbamic acid

A solution of 25 g rapamycin 42-p-nitrophenyl carbonate in 11 mlN,N-dimethylformamide was treated at room temperature under nitrogenwith 714 mg 1-aminopiperidine. The reaction mixture was stirred at roomtemperature for five hours. The solvent was removed by vacuum pump andthe residue was dissolved in 300 ml dichloromethane. The organicsolution was washed with water, dried with MgSO₄, evaporated, and theresidue chromatographed on silica gel. Elution withmethanol/dichloromethane (1.5/98.5) afforded 470 mg of the title productas a whim solid, mp 110°-115°.

IR (KBr): 3420 (OH and NH), 1715 (lactone and ketone C═O), 1640 (amideC═O), 1450, 1235, 1100, and 990 cm⁻¹.

¹ HNMR (CDCl3, 400 MHz): δ4.55 (m, 1H, C₄₂ -H), HERE 3.38, 3.33, 3.14(each s, 3H, OCH₃), 2.73 (m, 4H, protons a), 1.65 (m, 6H, protons b)ppm. ##STR19##

MS (neg. ion FAB): 1039.6 (M⁻), 590.4, 447.3.

Example 20

Rapamycin 42-ester with piperidine-1-carbamic acid hydrochloric acidsalt

A solution of 25/mg rapamycin 42-ester with piperidine-1-carbamic acidin a mixture of 1 ml ethyl acetate and 4 ml dry ether was treated at 0°under nitrogen with 0.36 ml of 1M HCl (gas) in ether. The hydrochloridesalt formed instantly. Stirring was continued at 0° under nitrogen for1/4 hour. The product was collected by filtration, washed with ether,and dried in vacuum to afford 130 mg of the title product as a whitesolid, mp 120°-125°.

IR (KBr): 3400 (OH and NH), 1735 (lactone and ketone C═O), 1640 (amideC═O), 1450, 1370, 1240, 1100, and 990 cm⁻¹.

¹ HNMR (DMSO-D6, 400 MHz): δ4.75 (m, 1H, C₄₂ -H), 3.26, 3.15, 3.04 (eachs, 3H. OCH₃) ppm.

MS (neg. ion FAB): 1039.5 (M⁻), 590.4, 447.3.

Example 21

Rapamycin 42-ester with [(3R)-(+)-3-aminopyrrolidine]carboxylic acid

A solution of 2.67 g rapamycin 42-p-nitrophenyl carbonate in 23 mldichloromethane was treated at 0° under nitrogen with 213 mg Of(3R)-(+)-3-aminopyrrolidine in 1 ml dichloromethane. The reactionmixture was stirred at room temperature under nitrogen for two hours,diluted with 300 ml ethyl acetate, washed with water, and dried withMgSO₄. The solvent was evaporated and the residue chromatographed onsilica gel. Elution with dichloromethane/methanol (94/6) afforded 1.1 gof the title product as a pale yellow solid, mp 100°-105°.

IR (KBr): 3420 (OH and NH), 1715 (lactone and ketone C═O), 1640 (amideC═O), 1450, 1420, 1190, 1100, and 990 cm⁻¹.

¹ HNMR (CDCl₃, 400 MHz): δ4.54 (m, 1H, C₄₂ -H), 3.50-3.60, (m, 5H,protons a), 3.39, 3.33, 3.14 (each s, 3H, OCH₃) ppm.

MS (neg. ion FAB): 1025.5 (M⁻), 590.3,433.3. ##STR20##

Example 22

Rapamycin 42-ester with [(3R)-(+)-3-aminopyrrolidine]carboxylic acidhydrochloric acid salt A solution of 190 mg of rapamycin 42-ester with[(3R)-(+)-3-aminopyrrolidine]carboxylic acid in a mixture of 6 ml ethylacetate and 6 ml dry ether was treated at 0° under nitrogen with 0.3 mlof 1M HCl (gas) in ether. The clear solution was evaporated to a volumeof about 4 ml and diluted with 15 ml ether. The crystallinehydrochloride salt formed, and was stirred at 0° under nitrogen for 1/4hour. The product was collected by filtration, washed with ether, anddried in vacuum to afford mg of the title product as a pale solid, mp147°-152° (dec).

IR (KBr): 3420 (OH and NH), 1715 (lactone and ketone C═O), 1640 (amideC═O), 1440, 1190, 1100, and 990 cm⁻¹.

¹ HNMR (DMSO-D₆, 400 MHz): a 4.35 (m, 1H, C₄₂ -H), 3.32, 3.15, 3.04(each s, 3H, OCH₃), 3.40-3.80 (m, 5H, protons a) ppm. ##STR21##C/H/N/analysis for C₅₇ H₈₉ N₃ O₁₄.H₂ O.HCl Calc.: 62.23/8.28/3.88; Found61.93/8.26/3.91.

Example 23

Rapamycin 42-ester with [(3S)-(-)-3-aminopyrrolidine]carboxylic acid

A solution of 3.0 rapamycin 42-p-nitrophenyl carbonate in 20 mldichloromethane was treated at 0° under nitrogen with 319 mg of(3S)-(-)-3aminopyrrolidine in 2 ml dichloromethane. The yellow solutionwas stirred at room temperature for 3 hours, diluted with 100 mldichloromethane, washed with water, and dried with MgSO₄. The solventwas evaporated and the residue chromatographed on silica gel. Elutionwith dichloromethane/methanol (93/7) afforded 1.32 g of the titleproduct as a pale white solid, mp 135°-140°.

IR (KBr): 3400 (OH and NH), 1715 (lactone and ketone C═O), 1640 (amideC═O), 1455, 1420, 1190, 1100 and 990 cm⁻¹.

¹ HNMR (CDCl₃, 400 MHz): δ4.55 (m, 1H, C₄₂ -H), 3.59, (m, 3H, protonsa), 3.20 (t. 2H, protons b), 3.39, 3.33, 3.14 (each s, 3H, OCH₃) ppm.

MS (neg. ion FAB): 1025.3 ##STR22## (M⁻), 590.2, 433.2.

Example 24

Rapamycin 42-ester with [(3S)-(-)-3-aminopyrrolidine]carboxylic acidsalt with hydrochloric acid

A solution of 700 mg of rapamycin 42-ester with[(3S)-(-)-3aminopyrrolidine]carboxylic acid in a mixture of 4 ml ethylacetate and 4 ml ether was treated at 0° under nitrogen with 0.75 ml of1M HCl (gas) in ether. The clear solution was diluted with 4 ml ether toinduce crystalline salt formation. Stirring was continued at 0° undernitrogen for 1/4 hour. The product was collected by filtration, washedwith ether, and dried in vacuum to afford 610 mg of the title product asa white solid, mp 136°-140° (dec).

IR (KBr): 3500 (OH and NH), 1715 (lactone and ketone C═O), 1640 (amideC═O), 1440, 1190, 1100, and 990 cm⁻¹.

¹ HNMR (DMSO-D₆, 400 MHz): δ8.13 (s, 2H, NH₂), 4.36 (m, 1H, C₄₂ -H),3.63-3.40 (m, 5H, protons a), 3.32, 3.15, 3.04 (each s, 3H, OCH₃) ppm.

MS (neg. ion FAB): 1025.6 (M⁻), 590.4, 433.3. ##STR23## C/H/N analysisfor C₅₇ H₈₉ N₃ O₁₄.H₂ O.HCl; Calc: 62.23/8.28/3.88; Found62.23/8.28/3.88; Found

Example 25

Rapamycin 42-ester with [1-(4-morpholinyl)propyl]carbamic acid

A solution of 9.0 g rapamycin 42 p-nitrophenyl carbonate in 50 mldichloromethane was treated at 0° under nitrogen atmosphere with 2.3 g4-(3-aminopropyl) morpholine in 4 ml dichloromethane. The reactionmixture was stirred at 0°/N₂ for two hours, diluted with 400 mldichloromethane, washed with water, and dried with MgSO₄. The solventwas evaporated and the residue chromatographed on silica gel. Elutionwith ethyl acetate/methanol (9/1 ) afforded 6.70 g of the title productas a white solid, mp 96°-99°.

IR (KBr): 3400 (OH and NH), 1745 (lactone and ketone C═O), 1650 (amideC═O), 1450, 1250, 1120 and 990 cm⁻¹.

¹ HNMR (CDCl₃, 400 MHz): δ4.54 (m, 1H, C₄₂ -H), 3.70, (t. 4H, protonsc), 3.37, 3.33, 3.14 (each s, 3H, OCH₃), 3.23 (t, 2H, protons b), 2.43(t, 6H, protons a) ppm. ##STR24## MS (neg. ion FAB): 1083.7 (M⁻), 590.4,491.3.

Example 26

Rapamycin 42-ester with [1-(4-morpholinyl)propyl]carbamic acid salt withhydrochloric acid

A solution of 530 mg rapamycin 42-ester with[1-(4-morpholinyl)propyl]carbamic acid in a mixture of 3.5 ml ethylacetate and 7.0 ml ether was treated at 0° under nitrogen with 0.5/ml 1MHCl (gas) in ether. The hydrochloride salt formed instantly. Stirringwas continued at 0° under nitrogen for 1/4 hour. The product wascollected by filtration, washed with ether, and dried in vacuum at roomtemperature to afford 490 mg of the title product as a white solid, mp106°-110° (dec).

IR (KBr): 3400 (OH and NH), 1715 (lactone and ketone C═O), 1645 (amideC═O), 1450, 1240, 1100, and 980 cm⁻¹.

¹ HNMR (DMSO-D₆, 400 MHz): δ4.37 (m, 1H, C₄₂ -H), 3.24, 3.12, 3.02 (eachs, 3H, OCH₃), 7.23 (s, 1H, NH) ppm.

MS (neg. ion FAB): 1083.3 (M⁻), 590.2, 491.2. C/H/N analysis for C₅₉ H₉₃N₃ O₁₅.HCl.H₂ O; Calc: 62.22/8.32/3.68; Found 61.98/8.49/3.43.

Example 27

Rapamycin 42-ester with (4-morpholinyl)propyl carbamic acid salt withmaleic acid

A solution of 552 mg rapamycin 42-ester with (4-morpholinyl) propylcarbamic acid in a mixture of 5 ml ethyl acetate and 5 ml ether wastreated at room temperature under nitrogen with 60 mg maleic acid in amixture or 1 ml ethyl acetate and 1 ml ether. The solvent was evaporatedleaving 550 mg title product as a white solid, mp 104°-107°.

IR (KBr): 3430 (OH and NH), 1725 (lactone and ketone C═O), 1650 (amideC═O), 1450, 1355, 1350, 1250, 1100, and 990 cm⁻¹.

¹ HNMR (DMSO-D₆): δ7.1 (s, 1H, --NH), 6.05 (s, 2H, maleate olefin), 4.39(m, 1H, C₄₂ -H), 3.27, 3.16, 3.05 (each s, 3H, OCH₃) ppm.

MS (neg. ion FAB): 1083.8 (M⁻), 590.5,491.5. C/H/N analysis for C₆₃ H₉₇N₃ O₁₉.2 H₁ O; Calc: 61.19/8.23/3.39; Found 60.95/8.05/3.32.

Example 28

Rapamycin 42-ester with (4-morpholinyl)propyl carbamic acid salt withcitric acid

A solution of 528 mg rapamycin 42-ester with (4-morpholinyl) propylcarbamic acid in a mixture of 2 ml ethyl acetate and 8 ml ether wastreated at room temperature under nitrogen with 96 mg citric acid in 1ml methanol. The resulting solution was stirred at room temperature for1/4 hour. The solution was evaporated to dryness to afford 620 mg of thetitle product as a white solid, mp 102°-106° (dec).

IR (KBr): 3420 (OH and NH), 1720 (lactone and ketone, acid C═O), 1650(amide C═O), 1450, 1250, 1100, and 990 cm⁻¹.

¹ HNMR (DMSO-D₆, 400 MHz): a 8.30 (s, 1H, NH⁺), 7.12 (t, 1H, NHCO--),4.38 (m, 1H, C₄₂ -H), 3.60 (t, 4H, --CH₂ --O--CH₂), 3.26, 3.14, 3.04(each s, 3H, OCH₃)ppm.

MS (Neg. ion FAB): 1083.6 (M⁻), 590.4, 491.4.

Example 29

Rapamycin 42-ester with (4-morpholinyl)propyl carbamic acid salt withmethanesulfonic acid

To a solution of 0.400 g (0.37 mmol) of the compound of Example 25 inether was added dropwise 8.1 ml (0.35 mmol) of 0.043M solution ofmethanesulphonic acid in ether. The resulting precipitate was collected.The solid was triturated with ether, filtered under a cone of nitrogen,and dried under vacuum to give 0.365 g (84%) of the title compound as awhite solid.

Calcd for C₅₉ H₉₃ N₃ O₁₅.CH₃ SO₃ H.3H₂ O: C, 58.37; H, 8.41; N, 3.40.Found: C, 58.68; H, 8.24; N, 3.47.

Example 30

Rapamycin 42-ester with (4-morpholinyl)propyl carbamic acid salt withphosphoric acid

To a solution of 0.500 g (0.461 mmol) of the compound of Example 25 inether was added dropwise 4.52 ml (0.438 mmol) of 0.102M solution ofphosphoric acid in methanol. The reaction mixture was stirred undernitrogen for about 30 minutes after which time the solvents were removedunder vacuum. The residue was triturated with ether, filtered under acone of nitrogen, and dried under vacuum to give 0.480 g (93% ) of thetitle compound as a cream colored solid.

Calcd for C₅₉ H₉₃ N₃ O₁₅.H₃ PO₄.4H₂ O: C, 56.51; H, 8.36; N, 3.35.Found: C, 56.62; H, 8.07; N, 3.40.

Example 31

Rapamycin 42-ester with (4-morpholinyl)propyl carbamic acid salt withL-monomethyl tataric acid

To a solution of 0.550 g (0.507 mmol) of the compound of Example 25 inether was added dropwise 7.57 ml (0.507 mmol) of 0.067M solution of L(+) tartaric acid monomethyl ester in methanol. The reaction mixture wasstirred under nitrogen for about 30 minutes after which time thesolvents were removed under vacuum. The residue was triturated withether, filtered under a cone of nitrogen, and dried under vacuum to give0.476 g (75%) of the title compound as a cream colored solid.

Calcd for C₅₉ H₉₃ N₃ O₁₅.C₅ H₈ O₆.1.5H₂ O: C, 60.26; H, 8.22; N, 3.29.Found: C, 60.05; H, 8.08; N, 3.27.

Example 32

Rapamycin 42-ester with (4-morpholinyl)propyl carbamic acid salt withL-tartaric acid

To a solution of 0.330 g (0.304 mmol) of the compound of Example 25 inether was added dropwise 2.89 ml (0.289 mmol) of 0.1M solution of L(+)tartaric acid in methanol. The reaction mixture was stirred undernitrogen for about 30 minutes after which time the solvents were removedunder vacuum. The residue was triturated with ether, filtered under acone of nitrogen, and dried under vacuum to give 0.307 g (86% ) of thetide compound as a cream colored solid.

Calcd for C₅₉ H₉₃ N₃ O₁₅.C₄ H₆ O₆.2H₂ O: C, 59.58; H, 8.18; N, 3.31.Found: C, 59.46; H, 8.30; N, 3.44.

Example 33

Rapamycin 42-ester with (4-morpholinyl)propyl carbamic acid salt withfurnaric acid

To a solution of 0.330 g (0.304 mmol) of the compound of Example 25 inether was added dropwise 2.89 ml (0.289 mmol) of 0.1M solution offumaric acid in methanol. The reaction mixture was stirred undernitrogen for about 30 minutes after which time the solvents were removedunder vacuum. The residue was triturated with ether, filtered under acone of nitrogen, and dried under vacuum to give 0.197 g (57%) of thetitle compound as a cream colored solid.

Calcd for C₅₉ H₉₃ N₃ O₁₅.C₄ H₄ O₄.2.5H₂ O: C, 60.78; H, 8.26; N, 3. 37.Found: C, 60.68; H, 8.05; N, 3.42.

Example 34

Rapamycin 42-ester with (4-morpholinyl)propyl carbamic acid salt withsulfuric acid

To a solution of 0.330 g (0.304 mmol) of the compound of Example 25 inether was added dropwise 1.60 ml (0.289 mmol) of 0.18M solution ofsulfuric acid in ether. The reaction mixture was stirred under nitrogenfor about 30 minutes after which time the solvents were removed undervacuum. The residue was triturated with ether, filtered under a cone ofnitrogen, and dried under vacuum to give 0.308 g (90%) of the titlecompound as a cream colored solid.

Calcd for C₅₉ H₉₃ N₃ O₁₅.H₂ SO₄.3H₂ O: C, 57.33; H, 8.23; N, 3.40.Found: C, 57.45; H, 8.13; N, 3.45.

Example 35

Rapamycin 42-ester with (4-morpholinyl)propyl carbamic acid salt withD-tartaric acid acid

To a solution of 0.330 g (0.304 mmol) of the compound of Example 25 inether was added dropwise 2.89 ml (0.289 mmol) of 0.1M solution of D(-)tartaric acid in methanol. The reaction mixture was stirred undernitrogen for about 30 minutes after which time the solvents were removedunder vacuum. The residue was triturated with ether, filtered under acone of nitrogen, and dried under vacuum to give 0.279 g (78%) of thetitle compound as a cream colored solid.

Calcd for C₅₉ H₉₃ N₃ O₁₅.C₄ H₆ O₆.25H₂ O: C, 59.16; H, 8.19:, N, 3.28Found: C, 59.16; H. 8.27; N, 3.10.

Example 36

Rapamycin 42-ester with [2-(1-morpholinyl)ethyl]carbamic acid

A solution of 1.6 g rapamycin 42-p-nitrophenyl carbonate in 10 mldichloromethane was treated at -10° under nitrogen atmosphere with 390mg 4-(2-aminoethyl)-morpholine in 2 ml dichloromethane. The reactionmixture was stirred at room temperature under N₂ for 13/4 hours, dilutedwith 200 ml dichloromethane, washed with water, and dried with MgSO₄.The solvent was evaporated and the residue chromatographed on silicagel. Elution with 2.5-3.0% methanol in dichloromethane afforded 0.88 gthe title product as a white solid, top. 110°-113°.

IR (KBr): 3400 (OH and NH), 1720 (lactone and ketone C═O), 1645 (amideC═O), 1450, 1110, and 985 cm⁻¹.

¹ HNMR (CDCl₃, 400 MHz): δ4.55 (m, 1H, C₄₂ -H), 3.70, (m, 4H, --O--CH₂--C--N--), 3.39, 3.33, 3.14 (each s, 3H, OCH₃), 2.46 (m, 6H, ##STR25##ppm

MS (neg. ion FAB): 1069.9 (M⁻), 590.5, 477.5.

Example 37

Rapamycin 42-ester with [2-(1-morpholinyl)ethyl]carbamic acid salt withhydrochloric acid

A solution of 550 mg rapamycin 42-ester with[2-(1-morpholinyl)ethyl]carbamic acid in 15 ml dry ether was treated at0° under N₂ with 0.76 ml 1M HCl (gas) in ether. The hydrochloride saltformed instantly. Stirring was continued at 0°/N₂ for 1/4 hour. Theproduct was collected by filtration, washed with ether, and dried invacuum to afford 450 mg of the title product as a white solid, mp110°-113°.

IR (KBr): 3400 (OH and NH), 1725 (lactone and ketone C═O), 1640 (amideC═O), 1450, 1250, 1100, and 988 cm⁻¹.

¹ HNMR (DMSO-D₆, 400 MHz): a 4.38 (m, 1H, C₄₂ -H), 3.97 (t, 4H, --CH₂--O--CH₂ --), 3.24, 3.13, 3.06 (each s, 3H, OCH₃) ppm.

MS (Neg. ion FAB): 1069.5 (M⁻), 590.3, 477.3.

Example 38

Rapamycin 42-ester with [(4-methylpiperazin-1-yl)propyl]carbamic acid

A solution of 1.0 g rapamycin 42-p-nitrophenyl carbonate in 15 mldichloromethane was treated at 0° under N₂ with 157 mg 1-(3-aminopropyl4-methylpiperazine in 1 ml dichloromethane. The reaction mixture wasstirred at room temperature under N₂ for 31/4 hours, diluted with 200 mldichloromethane, washed with water, and dried with MgSO₄. The solventwas evaporated and the residue chromatographed on silica gel. Elutionwith 7% methanol in dichloromethane afforded 470 mg the title product asa white foam, mp 100°-105°.

IR (KBr): 3430 (OH and NH), 1735 (lactone and ketone C═O), 1650 (amideC═O), 1530, 1465, 1385, 1260, 1115, and 1000 cm⁻¹.

¹ HNMR (DMSO-D₆, 400 MHz): δ4.53 (m, 1H, C₄₂ -H), 3.38, 3.33. 3.14 (eachs, 3H, OCH₃), 2.32 (s, 3H, ##STR26## ppm.

MS (Neg. ion FAB): 1096.6 (M⁻), 590.3, 504.4.

Example 39

Rapamycin 42-ester with [(4-methylpiperazin-1-yl)propyl]carbamic acidsalt with hydrochloric acid

A solution of 244 mg rapamycin 42-ester with[(4-methylpiperazin-1-yl)propyl]carbamic acid in a mixture of 4 ml ethylacetate and 6 ml dry ether was treated at 0° under N₂ with 0.33 ml 1MHCl (gas) in ether. The hydrochloride salt formed instantly. Stirringwas continued at 0°/N₂ for 1/4 hour. The product was collected byfiltration, washed with ether, and dried in vacuum to afford 158 mg ofthe title product as a white solid, mp 125°-130°.

IR (KBr): 3420 (OH and NH), 1725 (lactone and ketone C═O), 1650 (amideC═O), 1520, 1450, 1400, 1250, 1110, and 990 cm⁻¹.

¹ HNMR (DMSO-D₆, 400 MHz): δ4.37 (m, 1H, C₄₂ -H), 3.26, 3.14. 3.04 (eachs, 3H, OCH₃), 2.68 (s, 3H, ##STR27## ppm.

MS (Neg. ion FAB): 1096.6 (M⁻), 590.3, 504.4.

Example 40

Rapamycin 42-ester with (N,N-di-n-butylaminopropylycarbamic acid

A solution of 1.0 g of rapamycin 42-p-nitrophenylcarbonate in 15 mldichloromethane was treated at -10° under a nitrogen atmosphere with 360mg N,N-di-n-butyl-1,3-propylanediamine in 2 ml dichloromethane. Theyellow solution was stirred at 0° under a nitrogen atmosphere for 31/2hours. The reaction mixture was diluted with 200 ml dichloromethane,washed with water, dried with MgSO₄ and evaporated. The residue waschromatographed on silica gel. Elution with 5% methanol indichloromethane afforded 490 mg of the title compound as a white foam,mp. 100°-105°.

IR (KBr): 3450 (OH and NH), 1745 (lactone and ketone C═O), 1640 (amideC═O), 1450, 1340, 1250, 1100 and 990 cm⁻¹.

¹ HNMR (CDCl₃, 400 MHz): δ4.54 (m, 1H, C₄₂ -H), 3.38, 3.33. 3.13 (s, 3H,OCH₃), 0.933 (t, terminal CH₃ of butyl) ppm.

MS (Neg. ion FAB): 1125.6 (M⁻), 590.3, 299.3, 167.1.

Example 41

Rapamycin 42-ester with (N,N-di-n-butylaminopropyl)carbamic acid saltwith hydrochloric acid

A solution of 300 mg rapamycin 42-ester withN,N-di-n-butylaminopropyl)carbamic acid in a mixture of 1 ml ethylacetate and 4 ml ether was treated at -78° under N₂ with 0.8 ml 1M HCl(gas) in ether. The hydrochloride salt formed instantly. Stirring wascontinued at -78° under N₂ for 1/2 hour. The product was collected byfiltration, washed with ether, and dried in vacuum to afford 245 mg ofthe title product as a white solid, mp 115°-120°.

IR (KBr): 3400 (OH and NH), 1725 (lactone and ketone C═O), 1540, 1450,1400, 1250, 1000, and 990 cm⁻¹.

¹ HNMR (DMSO-D₆, 400 MHz): a 4.37 (m, 1H, C₄₂ -H), 3.04, 3.14. 3.35(each s, 3H, OCH₃), 0.90 (t, terminal CH₃ of butyl)ppm.

MS (Neg. ion FAB): 1125.6 (M⁻), 590.3, 533.4, 229.3, 167.1.

The following representative compounds were prepared from rapamycin42-p-nitrophenyl carbonate and the appropriate amine by employing themethod used to prepare the compound of Example 9.

Example 42

Rapamycin 42-ester with[2-(4-pyrimidin-2-yl-piperazin-1-yl)-ethyl]carbamic acid, m.p. 103°-106°C.

Example 43

Rapamycin 42-ester with [2-(morpholine-4-carbonyloxy)-ethyl]carbamicacid, m.p. 97°-100° C.

Example 44

Rapamycin 42-ester with{2-[2-(pyridin-2-yl-ethyl)carbamoyloxy]-ethyl}carbamic acid, m.p.89°-92° C.

Example 45

Rapamycin 42-ester with 4-(pyridin-2-yl)-piperazine- 1-carboxylic acid,m.p. 118°-132° C.

Example 46

Rapamycin 42-ester with [3-(imidazol-1-yl)propyl]-carbamic acid, m.p.103°-106° C.

Example 47

Rapamycin 42-ester with 4-benzyl-piperazine-1-carboxylic acid, m.p.111°-120° C.

Example 48

Rapamycin 42-ester with 4-(pyrimidin-2-yl)-piperazine-1-carboxylic acid,m.p. 117°-126° C.

Example 49

Rapamycin 42-ester with 4-phenyl-piperazine-1-carboxylic acid, m.p.119°-126° C.

Example 50

Rapamycin 42-ester with benzimidazole-1-carboxylic acid, m.p. 122°-133°C.

Example 51

Rapamycin 42-ester with [2-(1-methyl-pyrrolidin-2-yl)-ethyl]carbamicacid, m.p. 99°-102° C.

Example 52

Rapamycin 42-ester with (pyridin-2-ylmethylene)-hydrazine-carboxylicacid, m.p. 128°-132° C.

Example 53

Rapamycin 42-ester with [4-(2-hydroxyethyl)-piperazin-1-yl]-carbamicacid, m.p. 115°-120° C.

What is claimed is:
 1. A compound of the structure ##STR28## wherein R¹and R² are each, independently, hydrogen, or ##STR29## ##STR30## is anitrogen containing heterocycle that may be saturated, unsaturated, orpartially unsaturated, and may be optionally mono-, di-, or tri-substituted with a group selected from alkyl of 1-6 carbon atoms,arylalkyl of 7-10 carbon atoms, alkoxy of 1-6 carbon atoms, cyano, halo,hydroxy, nitro, carbalkoxy of 2-7 carbon atoms, trifluoromethyl, amino,dialkylamino of 1-6 carbon atoms per alkyl group, dialkylaminoalkyl of3-12 carbon atoms, hydroxyalkyl of 1-6 carbon atoms, alkoxyalkyl of 2-12carbon atoms, alkylthio of 1-6 carbon atoms, --SO₃ H, --PO₃ H, and --CO₂H;with the proviso that R¹ and R² are not both hydrogen; or apharmaceutically acceptable salt thereof.
 2. The compound according toclaim 1 wherein R¹ is ##STR31##
 3. The compound according to claim 2,wherein ##STR32## is an optionally substituted morpholinyl orpiperazinyl group.
 4. The compound of claim 1 which is rapamycin42-ester with 4-(3-hydroxypropyl) piperazine-1-carboxylic acid or apharmaceutically acceptable salt thereof.
 5. The compound of claim 1which is rapamycin 42-ester with morpholine-4-carboxylic acid or apharmaceutically acceptable salt thereof.
 6. The compound of claim 1which is rapamycin 42-ester with 4-methylpiperazine-1-carboxylic acid ora pharmaceutically acceptable salt thereof.
 7. The compound of claim 1which is rapamycin 42-ester with 4-methyl-piperazine-1-carboxylic acidsalt with hydrochloric acid.
 8. The compound of claim 1 which israpamycin 42-ester with piperazine-1-carboxylic acid or apharmaceutically acceptable salt thereof.
 9. The compound of claim 1which is rapamycin 42-ester with 4-(2-hydroxyethyl)piperazine-1-carboxylic acid or a pharmaceutically acceptable saltthereof.
 10. The compound of claim 1 which isa) rapamycin 42-ester with4-(pyridin-2-yl)-piperazine-1-carboxylic acid, b) rapamycin 42-esterwith 4-benzyl-piperazine-1-carboxylic acid, c) rapamycin 42-ester with4-(pyrimidin-2-yl)-piperazine-1-carboxylic acid, d) rapamycin 42-esterwith 4-phenyl-piperazine-1-carboxylic acid, e) rapamycin 42-ester withbenzimidazole-1-carboxylic acid, f) rapamycin 42-ester with[(3S)-(-)-3-aminopyrrolidine]carboxylic acid or a pharmaceuticallyacceptable salt thereof.
 11. The compound of claim 10 which is rapamycin42-ester with [(3S)-(-)-3-aminopyrrolidine]carboxylic acid salt withhydrochloric acid.
 12. The compound of claim 1 which is rapamycin42-ester with [(3R)-(+)-3-aminopyrrolidine]carboxylic acid or apharmaceutically acceptable salt thereof.
 13. The compound of claim 1which is rapamycin 42-ester with [(3R)-(+)-3-aminopyrrolidine]carboxylicacid hydrochloric acid salt.